High density electrophoresis device and method

ABSTRACT

The present invention provides, in one aspect, an apparatus for electrophoretic separation of analytes. In one aspect, the apparatus comprises a disc-shaped substrate defining (1) a central reservoir region, (2) a plurality of electrophoretic channels in fluid communication with, and emanating substantially radially from, the central reservoir region, the channels being coplanar with each other, and each channel having (i) a proximal end which is linked to the reservoir region, and (ii) a distal end, and preferably (3) for each channel, at least one chamber, and preferably three chambers, linked by a passageway in fluid communication with the distal end of that channel. Preferably, each passageway leads from each chamber in a direction that is initially away from the central reservoir region, whereby centrifugation of the substrate about a central axis that is perpendicular to the channels is effective to disperse liquid from the central reservoir region into the channels and chambers, such that any air bubbles in the chambers, channels, and passageways are forced towards the axis of rotation, when such liquid is present in the central reservoir region.

This application claims the benefit of priority under 35 U.S.C. 119(e)to prior application Ser. No. 60/144,103 filed Jul. 16, 1999, which isincorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to electrophoretic analysis of analytes ofinterest. More particularly, the invention relates to small-scaledevices for conducting electrophoretic separation and/or analysis ofanalytes, as well as chemical and biochemical methods employing suchdevices.

REFERENCES

Bergot et al., PCT Pub. No. WO 91/07507.

Eckstein, F., Oligonucleotides and Analogs: A Practical Approach,Chapters 8 and 9, IRL Press, Oxford, GB (1991).

Fodor, S. P .A., et al., U.S. Pat. No. 5,445,934 (1995).

Fung et al, U.S. Pat. No. 4,757,141.

Grossman, P. D., and J. C. Colburn (eds.), Capillary Electrophoresis:Theory and Practice, Academic Press, Inc., London, UK (1992).

Haugland, Handbook of Fluorescent Probes and Research Chemicals,Molecular Probes, Inc., Eugene, Oreg. (1992).

Hobbs, Jr., et al., U.S. Pat. No. 5,151,507.

Huang, X. C., et al., Anal. Chem. 64:967 (1992).

Jackson, P., PCT Pub. No. WO 91/05256.

Keller and Manak, DNA Probes, 2nd Ed., Stockton Press, New York (1993).

Kheterpal et al., Eletrophoresis 17:1852-1859 (1996).

Landegren et al., U.S. Pat. No. 4,988,617.

Lee et al., EP 805190 A2 (1997).

Livak et al., PCT App. No. PCT/US98/09657.

Madou, M., Fundamentals of Microfabrication, CRC Press, LLC, Boca Raton,Fla. (1997).

Mathies, R. A., et al., U.S. Pat. No. 5,091,652 (1992).

Matthews et al, Anal. Biochem. 169:1-25 (1988).

Menchen, S., et al., PCT Pub. No. WO 94/05688 (1994).

Menchen, S., et al., U.S. Pat. No. 5,188,934 (1993).

Pastinen, T., et al., Genome Res. 7:606-614 (1997).

Rosenblum et al., Nucl. Acids Res. 25:4500-4504 (1997).

Sze, S. M., ed., VLSI Technology, 2^(nd) Ed., McGraw-Hill Publishing,New York, N.Y. (1988).

Whiteley et al., U.S. Pat. No. 4,883,750.

BACKGROUND

The structural analysis of polynucleotides and other biomolecules isplaying an increasingly important role in modern molecular biology. Withthe advent of polynucleotide amplification technology, e.g., PCR, andprojects directed towards sequencing the human genome, the level ofinterest in this area is high. In particular, the need to process largenumbers of samples as quickly as possible has led to the need foranalytical systems with increased resolution, throughout, andautomation.

It would be desirable to have a device which permits efficient,large-scale analysis of many samples in as small an area as possible, inorder to reduce cost and the amount of sample manipulation. At the sametime, the device should provide reproducible, high sensitivity detectionof analytes of interest. Preferably, the device will be compatible witha variety of different sample types and will be amenable to re-use withdifferent sample sets.

SUMMARY

In one aspect, the present invention provides an apparatus forelectrophoretic separation of analytes. In a preferred embodiment, theapparatus comprises a planar substrate defining (1) a central reservoirregion, (2) a plurality of electrophoretic channels in fluidcommunication with, and emanating substantially radially from, thecentral reservoir region, the channels being coplanar with each other,and each channel having (i) a proximal end which is linked to thereservoir region, and (ii) a distal end. At the distal end of eachchannel, the substrate further defines at least one chamber linked influid communication with the distal end of the channel. For example,each channel can be linked to a sample chamber, a sample-receivingchamber, and a running buffer chamber. Alternatively, each channel canbe linked to two distal chambers. Each one or more chambers ispreferably linked to the distal end of a channel by a passageway thatleads from each chamber in a direction that is initially away from thecentral reservoir region, whereby centrifugation of the substrate abouta central axis that is perpendicular to the channels is effective todisperse liquid from the central reservoir region into the channels andchambers, such that any air bubbles in the chambers, channels, andpassageways are forced towards the axis of rotation, when such liquid ispresent in the central reservoir region.

The apparatus preferably includes electrodes for applying a voltagepotential between the chambers and the central reservoir. The apparatusmay also include a detector for detecting selected components which maybe present in the channels. In one embodiment, the detector andsubstrate are disposed such that the detector and/or substrate arerotatable relative to each other to permit rotary detection. Forexample, in one approach, the detector can be rotatable about a centralaxis within the central reservoir region, for detecting signal emissionfrom each of the channels at a selected distance from the axis, or alonga selected length of each channel. In an alternate embodiment, thesubstrate may be rotatable about a central axis such that the channelspass sequentially by the detector, for detecting one or more componentsthat may be present in the channels. In a preferred embodiment, thedetector is adapted for detecting a fluorescent or chemiluminescentsignal.

In one embodiment, the apparatus may include an annular septum thatcovers, and which may partially define, the chambers, and which permitsneedle access to the chambers for delivery of liquids to the chambers.

In another embodiment, one or more of the channels may contain anelectrophoresis medium, such as a covalently crosslinked medium, anoncovalently crosslinked medium, or a flowable medium.

In another aspect, the invention provides a method for preparing aplurality of electrophoretic paths which are substantially bubble-free.The method may include providing an apparatus such as described above,such that the reservoir region contains a liquid or is in fluidcommunication with a liquid source, and centrifuging the substrate abouta central axis that is perpendicular to the channels so that the liquidis dispersed from the central reservoir region into the channels andchambers, such that any air bubbles in the chambers, channels, and/orpassageways are forced towards the axis of rotation, yielding aplurality of bubble-free electrophoretic paths between the reservoir andthe chambers.

In an alternate embodiment, the method may include providing anapparatus such as described above such that the reservoir region, andoptionally the channels, passageways, and/or chambers, contain a liquid,and centrifuging the substrate about a central axis that isperpendicular to the channels so that the liquid is dispersed from thecentral reservoir region into the channels and chambers, such that anyair bubbles in the chambers, channels, and/or passageways are forcedtowards the axis of rotation, yielding a plurality of bubble-freeelectrophoretic paths between the reservoir and the chambers.

The apparatus and methods discussed above can also be used for sampleanalysis. In one aspect, the invention includes a method for analyzing aplurality of samples. The method preferably includes providing anapparatus such as describes above, such that the central reservoirregion, channels, and chambers contain a liquid medium suitable forelectrophoresis of such samples. Samples are provided in one or more ofthe sample chambers, and an electric field is applied under conditionseffective to cause migration of sample(s) through at least one channeltowards the central reservoir region. The channel(s) may be interrogatedbefore, during and/or after electrophoresis to detect one or more samplecomponents in the channel(s).

The invention may be applied to the separation and/or analysis of any ofa variety of samples, particularly proteins, nucleic acids,polysaccharides, small molecules, and the like. Also, sample componentsto be detected may be labeled with detectable labels, e.g., fluorescentor chemiluminescent labels, to aid detection. The invention is alsouseful in combination with a wide variety of sample preparation methods,such as the polymerase chain reaction, oligonucleotide ligation assays,restriction fragment analysis, polymer sequencing, screening assays, andthe like.

These and other features and aspects of the invention will be furtherunderstood in light of the following description and the accompanyingdrawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a cross-sectional overview of a substrate in accordancewith the invention;

FIG. 2 shows an enlarged view of the central reservoir of the substratefrom FIG. 1;

FIG. 3 shows an enlarged view of the distal end of a channel havinglinked by passageways to a sample chamber, a sample-receiving chamber,and a running buffer chamber;

FIGS. 4A-4C show exemplary configurations for providing electrodes tothe chambers and central reservoir to control electrical voltages andcurrents;

FIGS. 5, 6 and 7 illustrate an exploded perspective view,cross-sectional view, and perspective view, respectively, of a substrateassembly of the invention;

FIG. 8 illustrates a centrifugal device for introducing liquid into achannel array of the invention with liquids and for removing airbubbles;

FIG. 9 shows a rotary detector for detecting and/or monitoring samplecomponents in the channels;

FIGS. 10A-10C illustrate an embodiment in which electrical voltages areprovided to the substrate by a contact card;

FIGS. 11A-11B illustrate an embodiment in which electrical voltages areprovided to the substrate by brush contacts.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention is directed to devices, apparatus, and methodsuseful for rapidly and conveniently analyzing a plurality of samplesusing electrophoresis. In one aspect, the insertion provides radialchannel arrays having electrophoretic pathways which, when filled withan appropriate liquid, are substantially bubble-free. The invention thusprovides improved reliability in high throughput electrophoresisapplications.

As used herein, the terms “channel” and “microchannel” areinterchangeable.

I. Apparatus

Reference is made to FIGS. 1 through 7, which illustrate variousfeatures of a radial channel array in accordance with a preferredembodiment of the invention. With reference to FIG. 1, substrate 20defines a plurality of microchannels 22 which emanate from a centralreservoir region 24. Each microchannel includes a proximal end 22 a anda distal end 22 b. The channels define lines that intersect at a centralpoint or axis 24 a (FIG. 2) in the center of the array. The centralreservoir region 24 provides a holding area for an electrophoresisbuffer which is in fluid communication with the proximal ends. Thecentral reservoir can also be used to introduce an electrophoresismedium or wash fluid into the channels.

In the embodiment shown in FIG. 3, each microchannel 22 terminates atits distal end with three chambers 26 a, 26 b and 26 c which are linkedto the distal end by connecting passageways 28 a, 28 b and 28 c. Eachchamber is linked to the associated microchannel by a passageway thatconnects to a radially remote region of the chamber. In other words,each passageway leads from each chamber in a direction that is initiallyaway from the central reservoir region, to facilitate centrifugalremoval of bubbles from the pathways of electrophoresis. The passagewaysfrom each of chambers 25 a, 26 b and 26 c are preferably linked to forma T injector function 30, wherein passageways 28 a and 28 c each form aright angle with respect to distal microchannel end 22 b at junction 30.These chambers may be used for various purposes, such as a samplechamber, running buffer chamber, and sample-receiving chamber,respectively, as discussed further below. For example, when chambers 26a and 26 c are used as a sample chamber and sample-receiving chamber, anelectric field between these two chambers can be used to draw a selectedsample volume into junction 30, for subsequent electrophoresis towardsthe central reservoir region. The chambers may also be provided with independently controllable electrodes for controlling electrical voltagesand currents in the device for various operations, such as electrodes 32a, 32 b, and 32 c shown in FIGS. 4A and 4B, and central electrode 32 din FIG. 4C.

Preferably, the three chambers 26 a, 26 b and 26 c associated with eachdistal end 22 b are located at different radial distances from thecenter of the substrate, to allow increased packing density of themicrochannels and chambers. Thus, in FIG. 3, it can be seen that chamber26 a is closest to the substrate center, followed by chamber 26 c, andthen 26 b, although other arrangements can also be used.

FIGS. 4A and 4B show a partial cross-sectional view, and partialoverhead view, respectively, of a substrate 20 that includes chambers 26a, 206 b and 26 c, and electrically conductive leads (electrodes) 32 a,32 b, and 32 c which are disposed along the surface of the substrate andwhich may extend into each chamber as shown. Also shown are optionalconcentric ring contacts 34 a, 34 b, and 34 c located on the other sideof the substrate, which may be electrically linked to leads 32 a, 32 b,and 32 c, respectively via connections 33 a, 33 b, and 33 c as shown.The concentric ring contacts can be included to perform elecrophoreticseparations in the channels in parallel. The chambers can be coveredwith an annular cover or septum 50 as discussed further with referenceto FIGS. 5-7 below.

FIG. 4C shows a partial cross-sectional view of the central region ofthe substrate, including cover layer 40, central reservoir region 24,and a threaded fastener 38 by which the substrate can be connected to amotor shaft 39 to rotate the substrate about central axis 24 a. Themotor shaft is preferably electrically grounded to provide theequivalent of a fourth electrode 32 d that can be used in combinationwith electrodes 32 a, 32 b, and 32 c, which permit directed movement ofcharged species between chambers or into and through the channels. Eachelectrode can be connected to an independently controllable voltagesource in order to control the movement of materials in the chambers andchannels at appropriate times.

Although FIGS. 4A-4C show particular electrode configurations, it willbe appreciated that any of a variety of other configurations can also beused. For example, the electrodes can be provided from above thechambers, e.g., as part of a cover layer bonded over the substrate inwhich the channels, passageways, chambers, and central reservoir aredefined. Similarly, the electrode in the central reservoir can bedisposed above the central reservoir, rather than through the bottom asshown at 32 d in FIG. 4C.

FIGS. 5-7 show an exemplary substrate assembly 100 which includes asubstrate 20, a channel cover 40, and an annular chamber cover 50.Channel cover 40 can be used to cover the channel array prior to fillingthe array with liquid media. Cover 40 may additionally include an inlet42 for introducing liquid into the central reservoir of the array, fordispersal into the channels. Annular chamber cover 50 is provided tocover the chambers during or after being filled with liquid.

The various features of the channel array can have any dimensions andconfigurations that are compatible with the utilities of the invention.Smaller dimensions are generally preferred in order to maximize thedensity of microchannels, to facilitate high sample throughout. Forexample, the microchannels can have any of a variety of cross-sectionalconfigurations, such as square, rectangular, semicircular, circular,concave, or V-shaped, with a broad range of widths and depths. Inparticular, the substrate may include discrete capillary tubes asmicrochannels disposed upon a planar surface of the substrate.Conveniently, the channels have rectangular, square, or concavecross-sections with depths and widths usually from about 250 μm to 1 μm,more typically from 100 μm to 1 μm, and preferably 50 μm or less.Similar considerations apply to the cross-sections of the passagewayswhich link the chambers to the distal ends of the channels.

The lengths of the channels are selected to permit a desired degree ofseparation of sample components, with shorter lengths providing shorterelectrophoresis times at the expense of decreased separation, and longerlengths providing longer separation paths and greater separation at theexpense of longer electrophoresis times. For example, channels of from 1cm to 50 cm lengths are suitable for many separations, although longerand shorter lengths can be used as well.

The chambers at the distal ends of the microchannels can have anyconfiguration such as circular, oval, square, and the like, and aretypically circular. The sizes and configurations of the chambers linkedto each microchannel can be the same or different. For example, thesample chamber should be large enough to receive a sufficient samplevolume, typically 10 μL or less. More generally, it is the preferredthat all of the chambers be large enough to contain a sufficient amountof buffer to avoid buffer depletion during electrophoresis.

The electrodes for generating electrical currents can be made of anysuitable conductive material, and are typically made from one or moremetals or alloys. Exemplary electrode materials include copper, silver,platinum, palladium, carbon, nichrome, and gold. The electrode materialscan be formed by known methods, conveniently by vapor deposition, silkscreen imprint, or other patterning techniques. The electrode materialsmay be coated with appropriate coating materials to inhibitelectrochemical reactions with samples and reagents. For example,electrodes may be coated with a permeation layer having a low molecularweight cutoff that allows passage of small ions but not reagent oranalyte molecules, as described, for example, in PCT Publ. No. WO95/12808 and WO 96/01836.

The passageways leading from the chambers to the channels are preferablyof minimum length to facilitate rapid electrophoresis. However, longerthan minimum lengths may be useful to help avoid leakage of liquid fromthe chambers into the channels.

The substrate defining the channel array is preferably weighted evenlyabout its central axis to allow stable centrifugation. Typically, thesubstrate is provided in the shape of a disc having a substantiallycircular perimeter.

The substrate can be formed from any material, or combination ofmaterials, suitable for the purposes of the invention. Materials whichmay be used will include various plastic polymers and copolymers, suchas polypropylenes, polystyrenes, polyimides, and polycarbonates.Inorganic materials such as glass and silicon are also useful. Siliconis advantageous in view so its compatibility with microfabricationtechniques and its high thermal conductivity, which facilitates rapidheating and cooling of the device if necessary.

The channel array may be formed by any suitable methodology available inthe art. For plastic materials, injection molding will generally besuitable to form channels, etc., having a desired configuration. Forsilicon, standard etching techniques from the semiconductor industry maybe used, as described in Madou (1997) and Sze (1988), for example.Etching techniques may be preferred for channel arrays with especiallysmall dimensions.

The substrate typically contains two or more laminated layers. Forexample, the channel array can be formed by etching or injection moldinginto the surface of a substrate, after which the channel array is sealedby overlaying a layer of material which covers at least the channels,passageways, chambers, and optionally the central reservoir, to preventevaporation of liquids from the array (see FIGS. 5-7).

In general, the substrate layers can be sealably bonded in a number ofways. Conventionally, a suitable bonding substance, such as a glue orepoxy-type resin, is applied to one or both opposing surfaces that willbe bonded together. The bonding substance may be applied to the entiretyof either surface, so that the bonding substance (after curing) willcome into contact with the chambers and/or channels. In this case, thebonding substance is selected to be compatible with the sample and anydetection reagents used in the assay. Alternatively, the bondingsubstance may be applied around the channel array so that contact withthe sample will be minimal or avoided entirely. The bonding substancemay also be provided as part of an adhesive-backed tape or membranewhich is then brought into contact with the opposing surface. In yetanother approach, the sealable bonding is accomplished using an adhesivegasket layer which is placed between the two substrate layers. In any ofthese approaches, bonding may be accomplished by any suitable method,including pressure-sealing, ultrasonic welding, and heat curing, forexample.

The substrates and apparatus of the invention may be adapted to allowrapid heating and cooling of the chambers and channels to facilitatesample preparation e.g., or PCR) and/or sample separation. In oneembodiment, the device is heated or cooled using an externaltemperature-controller. The temperature-controller is adapted toheat/cool one or more surfaces of the device, or may be adapted toselectively heat the detection chambers themselves. To facilitateheating or cooling with this embodiment, the substrate material ispreferably formed of a material which has high thermal conductivity,such as copper, aluminum, or silicon. Alternatively, a substrate layerin contact with the chambers and/or channels may be formed from amaterial having moderate or low thermal conductivity, such that thetemperature of the all or selected chambers and/or channels can beconveniently controlled by heating or cooling the heat-conductive layerregardless of the thermal conductivity of other layers in the substrate.In one preferred embodiment, an outer layer is provided across one ofthe surfaces of substrate as an adhesive copper-backed tape.

In an alternative embodiment, means for modulating the temperature ofthe detection chambers is provided in the substrate of the deviceitself. For example, the substrate may include resistive traces whichcontact regions adjacent the sample chambers, whereby passage ofelectrical current through the traces is effective to heat or cool thechambers. This approach is particularly suitable for silicon-basedsubstrates, and can provide superior temperature control.

For optical detection, the material defining the channel array ispreferably optically transparent or at least includes transparentregions or windows which permit viewing of part or all of each channel,and optionally permit viewing of the chambers, passageways, and/or otherelements of the channel array. For this purpose, silica-based glasses,quartz, polycarbonate, or an optically transparent plastic layer may beused, for example. Selection of the particular transparent material willdepend in part on the optical properties of the material and thespectroscopic characteristics of the signal to be detected. For example,in a fluorescence-based assay, the material should have low fluorescenceemission at the wavelength(s) being measured. The window material shouldalso exhibit minimal light absorption for the signal wavelengths ofinterest.

Other layers or materials may also be included. For example, the samplechamber may be lined with a material that has high heat conductivity,such as silicon or a heat-conducting metal, to permit rapid heating andcooling of the sample. Silicon surfaces which contact the sample arepreferably coated with an oxidation layer or other suitable coating, torender the surface more inert. Similarly, where a heat-conducting metalis used in the substrate, the metal can be coated with an inertmaterial, such as a plastic polymer, to prevent corrosion of the metaland to separate the metal surface from contact with the sample.

For electrophoresis of samples, the channel array is preferably filledwith an electrophoresis medium via the central reservoir region. Forthis purpose, the central reservoir region and channels may be enclosedusing a cover equipped with an inlet, for transporting liquid into thearray, and the distal chambers can be covered with an annular cover,such as cover 50 in FIG. 5.

In one embodiment, the annular cover is porous to air but is relativelyimpervious to aqueous liquid. Thus, with reference to FIG. 5, liquidintroduced through inlet 42, e.g., by pressure or by centrifugal force,flows through the radial channels and into the distal chambers such thatdisplaced air escapes through the annular cover. Once the chambers arefull, the porous cover provides back pressure sufficient to prevent theliquid from leaking out of the chambers. The porous annular cover maythen be replaced with an annular septum to seal the chambers but allowintroduction of fluids to the channels by cannula or needle. In analternate approach, an annular cover, which may be porous or not, isplaced in close (but not sealed) contact with the outer radial region ofthe substrate during filling, so that excess liquid escapes through anarrow gap between the annular surface and outer substrate surface.After filling is complete, the annular ring can be pressed securelyagainst the opposing substrate surface to seal the chambers, such thatexcess liquid between the annular ring and substrate surfaces issqueezed out. Filling can be promoted further by placing the substrateassembly in a vacuum atmosphere, to help reduce resistance from any airoccupying the channels and chambers.

According to one aspect of the invention, filling the channel withliquid can be facilitated by spinning the array about the central axisperpendicular to the array plane, to drive fluid towards the peripheryof the array by centrifugal force. In addition, any bubbles in thechambers will be driven towards the center of the array, away from thepassageways linking the chambers to the channels. In this regard, FIG. 8shows a substrate assembly 100 seated in a centrifugation device 200,for centrifuging the assembly as just described. The substrate assembly100 is spun at a speed and for a time sufficient to remove substantiallyall bubbles from the channels and passageways, to provide continuouselectrical and liquid pathways between the central reservoir and thechamber electrodes.

The electrophoresis medium in the channels can be any medium deemedappropriate by the user for the purposes of this invention. Usually, themedium will be an aqueous medium, although nonaqueous media are alsocontemplated. Additionally, the medium may contain agents that impede orotherwise alter the migration rates of sample components. Examples ofsuch agents include water-soluble polymers such as agarose,polyacrylamide, polymethacrylamide, methyl cellulose,hydroxyethylcellulose, hydroxypropylcellulose,hydroxypropylmethylcellulose, polyethylene glycol, galactomannan,polyvinyl alcohol, polyacryloylaminoethoxyethanol, polyethylene imine,polyvinylacetate, polyvinylpyrrolidone, and polyvinyloxazolidone, andalso fluorine-containing polymers (e.g., see Ramakrishna et al., U.S.Pat. Nos. 5,552,028 and 5,567,292; Grossman, U.S. Pat. No. 5,374,527;Menchen et al., U.S. Pat. No. 5,468,365; and Grossman et al. (1992)).The foregoing materials can be used to form entangled matrices ifconcentrations are sufficiently high, although more dilute(non-entangled) concentrations may also be used. Covalently crosslinkedmedia, such as polyacrylamide crosslinked with bis-acrylamide, can alsobe used, in which case loading is typically accomplished before themedium is crosslinked, e.g., by UV irradiation or by adding an initiatorreagent such as tetramethylenediamine plus ammonium persulfate.

If desired, the inner surfaces of all or part of the chananel array canbe coated with any suitable coating material, to reduce sampleadsorption. Since electrophoresis is usually performed in an aqueousseparation medium, adsorption of sample can usually be reduced bycovering the inner surfaces of the separation cavity with a hydrophiliccoating that masks potentially adsorptive surface regions. Exemplaryreagents for coating adsorptive surfaces include poyacrylamide,polymethacrylamide, polyvinyl alcohol, polyethers, cellulose acetate,polyalkylene oxides, poly(vinyl pyrrolidone), and other materials as areknown in the art. Preferably, such coatings are attached to interiorsurfaces covalently, although adsorptive noncovalent coatings may alsobe suitable.

Coating reagents for reducing sample adsorption can also be used tocontrol the magnitude of electroosmotic flow (EOF). For example, EOFalong glass silicate surfaces can be substantially reduced by coatingthem with a neutral reagent that masks a substantial percentage ofsurface silanol groups. The magnitude of EOF can be further controlledby using coating reagents that include positively or negatively chargedgroups. Positively charged coatings can be used to nullify surfacenegative charges to give a net surface charge of zero, so that EOF=0.Coatings with higher positive charge densities can be used to reversethe direction of EOF for charged surface materials. This can be usefulfor slowing the net migration rates of positively charged samplespecies. Conversely, negatively charged coatings can be used to impartto or increase the magnitude of negative charge on surfaces, to slow thenet migration rates of negatively charged species. Representativepositively charged coatings include polyethyleneimine, quaternizedpolyethyleneimine, and chitosans, for example. Representative negativelycharged coatings include carboxylate and sulfonate containing materials,such as poly(methylglutamate) and 2-acrylamido-2-methylpropanesulfonatepolymers, for example. It will be recognized that charged coatings canalso effectively reduce sample adsorption, especially for samples havingthe same charge polarity as the coating (e.g., Wiktorowicz, U.S. Pat.Nos. 5,015,350 and 5,181,999).

The choice additives, if preset in the separation medium will depend inpart on the sample and the nature of the interior surfaces, as well asother factors. In some applications, it may be desirable to use both acovalent surface coating and soluble buffer agents to control sampleadsorption and EOF.

Samples may be from any source which can be dissolved or extracted intoa liquid that is compatible with the uses of present invention, andwhich may potentially contain one or more analytes of interest. Forexample, the sample may be a biological fluid such as blood, serum,plasma, urine, sweat, tear fluid, semen, saliva, cerebral spinal fluid,or a purified or modified derivative thereof. Amples may also beobtained from plants, animal tissues, cellular lysates, cell cultures,microbial samples, and soil samples, for example. The sample may bepurified or pre-treated if necessary before testing, to removesubstances that might otherwise interfere with analyte detection.Typically, the sample fluid will be an aqueous solution, particularlyfor polar analytes such as polypeptides, polynucleotides, and salts, forexample. The solution may include surfactants or detergents to improveanalyte solubility. For non-polar and hydrophobic analytes, organicsolvents may be more suitable.

For each channel, sample is preferably loaded into a distal chamber,referred to herein as a sample chamber, by injection through a chamberwall, e.g., via a septum material such as discussed above. Pre-existingair and/or liquid in the chamber is preferably allowed to escape thechamber via a second needle of cannula which passes through the chamberwall, so that the chamber is preferably uniformly loaded with thesample. One or more of the other distal chambers of each channel canalso be loaded with a selected liquid medium using the same loadingtechnique. Sample loading can be automated using a roboticallycontrolled sample dispensor, if desired.

After sample, loading is complete, the substrate may be centrifuged asdiscussed above with respect to FIG. 7, in order to drive any airbubbles towards the center of the channel array, and out of the variouspaths of electrophoresis.

Once loaded, an aliquot of sample is preferably transferred from thesample chamber to the distal end of the channel by applying an electricfield between the sample-containing chamber and a selected “sink”(waste) chamber. For example, with reference to FIG. 3, sample inchamber 25 a can be transferred electrokinetically into junction 30 byapplying an electric field between chambers 26 a and 26 c. The amount ofsample (sample plug) transferred into the pathway of channel 22 isproportional to the cross-sections of passageways 28 a and 28 c atjunction 30 (which defines the initial band width of the aliquot) and bythe cross-section of channel 22 at junction 30. After the first electricfield is shut off, an electric field is applied between chamber 26 b andcentral reservoir 24, thereby drawing the sample plug into channel 22and initiating separation of sample components on the basis of differentelectrophoretic mobilities. Any other appropriate sample loadingsequence may also be used.

Electrophoretic operations can be carried out in the channelssimultaneously (in parallel), individually (sequentially), or anycombination thereof. With reference to FIGS. 4A-4C discussed above,electrophoresis can be performed sequentially by applying appropriatevoltages to electrodes 32 a, 32 b, 32 c, and 32 d (without needing rings34 a-34 c and connectors 33 a -33 c). Conveniently, this can beaccomplished by attaching an electrical contact card to the upper orlower surface of the substrate, such that the contact card hasindividual electrical contacts that align with the substrate electrodes,as illustrated in FIGS. 10A-10C.

FIGS. 10A-10C show a cross-sectional side view and overhead view of anexemplary contact card 400 which can be used to supply separateelectrical voltages to the distal chambers of a microchannel. Contactcard 400 includes upper and lower protrusions 402 a and 402 b whichdefine a cavity therebetween, for snugly gripping an electrical lead onthe substrate, such as electrical leads 32 a, 32 b and 32 c (FIGS. 4Band 10C). The upper and lower protrusions of the card are preferablymade of a flexible material so that the contact card can be easilyclamped onto and removed from substrate 20. Contact card 400 alsoincludes electrical leads 404 a, 404 b, and 404 c having exposedterminal ends 406 a, 406 b, and 406 c, respectively, for contactingelectrical leads 32 a, 32 b, and 32 c, as illustrated in cross-sectionalside view in FIG. 10C.

Simultaneous electrophoretic operations can be performed conveniently byapplying appropriate voltages to one or more conductive rings which areelectrically connected to the electrodes in the distal chambers, such asrings 34 a, 34 b and 34 c shown in FIGS. 4A and 4B. For this embodiment,the voltage potentials are preferably provided through conductivebrushes which can remain in contact with the concentric rings while thesubstrate is rotated about its axis for analyte detection, if desired.For example, with reference to FIGS. 11A and 11B, a substrate 20 havingconcentric ring contacts such as contacts 34 a, 34 b, and 34 c shown inFIG. 4A, is contacted with corresponding brush contacts 502 a, 502 b,and 502 c which are held by one or more holders, such as holders 504 a,504 b, and 504 c.

Simultaneous electrophoresis has the advantage of faster sampleanalysis. Sequential electrophoresis, on the other hand, allows morecareful control of electrophoresis conditions in each channel.

Sample components of interest may be detected in the channels by any ofa variety of techniques, such as fluorescence detection,chemiluminescence detection, UV-visible adsorption, radioisotopedetection, electrochemical detection, and biosensors, for example. Foroptically based detection methods (e.g., fluorescence, absorbance, orchemiluminescence), the substrate assembly should contain at least onedetection zone near the proximal end of each channel.

Typically, optical detection is performed from above or below the planeof the substrate assembly. In general, optical signals to be detectedwill involve absorbance or emission of light having a wavelength betweenabout 180 nm (ultraviolet) and about 50 μm (far infrared) Moretypically, the wavelength is between about 200 nm (ultraviolet) andabout 800 nm (near infrared). For fluorescence detection, any opaquesubstrate material in the zone of detection preferably exhibits lowreflectance properties so that reflection of the illuminating light backtowards the detector is minimized. Conversely, a high reflectance willbe desirable for detection based on light absorption. Withchemiluminescence detection, where light of a distinctive wavelength istypically generated without illuminating the sample with an outsidelight source, the absorotive and reflective properties of the substrateassembly will be less important, provided that at least one opticallytransparent window is present per channel for detecting the signal.Preferably, substantially all of the substrate assembly is transparent,to allow visualization of the entire channel array.

When the material defining the upper surface and sides of the channelsare optically clear, and detection involves fluorescence measurement,the channels can be illuminated with excitation light through the sidesof the channels (parallel to the plane of the substrate assembly), ormore typically, diagonally from above (e.g., at a 45 degree angle), andemitted light is collected from above the substrate assembly, usually ina direction perpendicular to the plane of the channel array.

FIG. 9 shows an exemplary detection system 300 comprising a rosary plate302, substrate assembly 100, and detector arm 304. Detector arm 304carries a detector rod 305 having a lower end that is positioned over aselected detection zone on the channel array of assembly 100. Inoperation, detector rod 306 is positioned over a detection zone of achannel for a time (or times) sufficient to collect a signal from thechannel to identify the presence of, and/or quantify, one or more samplecomponents in the channel.

In one approach, the detector rod remains over the detection zone of thechannel during electrophoretic separation of the sample, to record anelectropherogram of components continuously or at discrete time pointsas they migrate past the detector rod. After the desired information hasbeen collected, assembly 100 is rotated so that the detector ispositioned over the detection zone in the next channel, and anotherelectropherogram is recorded. Thus, electrophoresis is performedsequentially from channel to channel, until the desiredelectropherograms have been obtained.

In another approach, signals are measured periodically in each channelduring simultaneous electrophoresis in two or more channels, by rotatingthe assembly at selected time intervals to collect electropherogramssimultaneously as a series of time points. Preferably, the frequency ofdata collection from each channel is sufficient to ensure collection ofat least two points, and preferably more, per component peak, tofacilitate accuracy and sensitivity of detection.

The sample components or analytes to be measured can be labeled tofacilitate sensitive and accurate detection. Labels may be direct labelswhich themselves are detectable or indirect labels which are detectablein combination with other agents. Exemplary direct labels include butare not limited to fluorophores, chromophores, (e.g. ³²P, ³⁵S, ³H)spin-labels, chemiluminescent labels (e.g., dioxetane-producingmoieties), radioisotopes, and the like. Exemplary indirect labelsinclude enzymes which catalyze a signal-producing event, and ligandssuch as an antigen or biotin which can bind specifically with highaffinity to a detectable anti-ligand, such as a labeled antibody oravidin. Many references on labeling molecules of interest, such as DNA,proteins, polysaccharides, and the like, are available. Exemplaryreferences include Matthews et al. (1988), Haugland (1992), Keller andManak (1993), Eckstein (1991), Fung et al.; Hobbs et al., Lee et al.,Menchen et al., Bergot et al., Rosenblum et al. (1997), and Jackson (WO91/05256).

In one preferred embodiment, sample components or target analytes aremeasured by fluorescence detection. To perform such detection, thedetection zone of each channel can be illuminated by a suitable lightsource, e.g. a high intensity mercury vapor lamp, laser, or the like.Preferably the illumination means is a laser having an illumination beamat a wavelength between 488 and 550 nm. More preferably, particularlyfor dye-labeled polynucleotides, illumination is accomplished using alaser light generated by an argon ion laser, particularly the 488 and514 nm emission lines of an argon ion laser, or the 532 nm emission lineof a neodymium solid-state YAG laser. Several argon ion lasers areavailable commercially which lase simultaneously at these lines, e.g.Cyonics, Ltd. (Sunnyvale, Calif.) Model 2001, or the like. Thefluorescence is then detected by a light-sensitive detector, e.g. , aphotomultipier tube, a charged coupled device, or the like.Conveniently, the fluorescence detector has a confocal arrangement, suchas described in Huang et al., 1992, Kheterpal et al., (1995) and otherreferences (see also Fodor, 1995, and Mathies et al., 1992).

Sample component signals can also be collected from one or more channelssimultaneously using an area-type detector, such as a charge-coupleddetector (CCD), (e.g., Model TE/CCD512SF, Princeton Instruments,Trenton, N.J.) with suitable optics (Ploem, 1993), such as described inYershov et al. (1996), or may be imaged by TV monitoring (Khrapko,1991). For radioactive signals (e.g., ³²P), a phosphorimager device canbe used (Johnston et al., 1990; Drmanac et al , 1992; 1993). Othercommercial suppliers of imaging instruments include General ScanningInc. (Watertown, Mass., www.genscan.com), Genix Technologies (Waterloo,Ontario, Canada; www.confocal.com), and Applied Precision Inc.

III. Utility

The present invention can be used for any of a wide variety ofapplications. The invention can be used for medical or veterinarypurposes, such as detecting pathogens, diagnosing or monitoring disease,genetic screening, determining antibody or antigen titers, detecting andmonitoring changes in health, and monitoring drug therapy. The inventionis also useful in a wide variety of forensic, environmental, andindustrial applications, including screening molecules for selectedactivities.

For example, the invention can be used to analyze varoius nucleotide andpolynucleotide analytes produced by a variety of techniques, such as thepolymerase chain reaction, oligonucleotide ligation assay (e.g.,Whiteley, et al. and Landegren et al.), minisequencing (Pastinen, etal., 1997), microsatellite/variable number or tandem repeat (VNTR)analyses (e.g., Livak et al.), restriction fragment length polymorphism.(RFLP) analysis, and Sanger-type sequencing (e.g., Lee et al., EP 805190A2, pp. 38-39)

The invention is also useful for analyzing other types of samplecomponents, such as polypeptides, amino acids, polysaccharides,monosaccharides, metabolites, drugs, etc. The invention is also usefulfor high-throughput screening, wherein a large number of differentmolecules are tested for a selected activity, such as binding of aligand to a receptor, activation or inhibition of an enzyme, and thelike.

More generally, the present invention provides a convenient way torapidly analyze analytes in a plurality of samples. The invention ishighly flexible in its applications, being adaptable to analysis of awide variety of analytes and sample materials. Furthermore, for arrayconfigurations in which distal chambers are linked to the channels bypassageways leading away from the center of the array, the inventionallows bubbles to be removed from electrophoretic paths bycentrifugation, prior to sample separation and analysis, therebyenhancing precision, accuracy and reproducibility of analyses. Moreover,very small volumes of sample are required since the dimensions ofchannel arrays of the invention can be very small.

While the invention has been described with reference to certainembodiments and examples, it will be appreciated that variousmodifications and variations can be made without departing from thespirit of the invention.

What is claimed is:
 1. Apparatus for electrophoretic separation ofanalytes, the device comprising: a substrate including (1) a centralreservoir region, (2) a plurality or electrophoretic channels in fluidcommunication with, and emanating substantially radially from, thecentral reservoir region, the channels being coplanar with each other,and each channel having (i) a proximal end which is linked to thecentral reservoir region, and (ii) a distal end, and (3) for eachchannel one or more chambers that are each linked by a passageway influid communication with the distal end of that channel, wherein eachpassageway leads from each chamber in a direction that is initially awayfrom the central reservoir region, whereby centrifugation of thesubstrate about a central axis that is perpendicular to the plane of thechannels is effective to disperse liquid from the central reservoirregion into said channels and chambers such that any air bubbles in thechambers, channels, and passageways are forced towards the axis ofrotation, when such liquid is present in the central reservoir region.2. The apparatus of claim 1, wherein the distal end of each channel islinked by passageways to two chambers.
 3. The apparatus of claim 1,wherein the distal end of each channel is linked by passageways to asample chamber, a sample-receiving chamber, and a running bufferchamber.
 4. The apparatus of claim 1, which further includes electrodesor applying a voltage potential between said chambers and the centralreservoir region.
 5. The apparatus of claim 1, which further includes adetector for detecting selected components which may be present in oneor more of the channels.
 6. The apparatus of claim 5, wherein thedetector is a fluorescence or chemiluminescence detector.
 7. Theapparatus of claim 5, wherein the detector is rotatable about thecentral axis within the central reservoir region, for detecting signalemission from each of the channels at a selected distance from the axis.8. The apparatus of claim 5, which further includes a mechanism forrotating the substrate about the central axis such that the channelspass sequentially by the detector, for detecting one or more componentsthat may be present in the channels.
 9. The apparatus of claim 1,wherein said chambers are defined in part by an annular septum thatcovers the chambers and permits needle-access to the chambers fordelivery of liquid to the chambers.
 10. The apparatus of claim 1,wherein at least one of said channels contains an electrophoresismedium.
 11. The apparatus of claim 10, wherein the electrophoresismedium is a flowable medium.
 12. The apparatus of claim 10, wherein theelectrophoresis medium is a covalently crosslinked medium.
 13. Theapparatus of claim 1, wherein the substrate defines at least 20 of saidchannels.
 14. The apparatus of claim 1, wherein said channels havecross-sectional diameters between 1 and 100 μm.
 15. The apparatus ofclaim 12, wherein said channels have cross-sectional diameters between 2and 50 μm.
 16. A method for preparing a plurality of electrophoreticpaths which are substantially bubble-free, comprising providing anapparatus as defined in claim 1 such that the reservoir region eithercontains a liquid or is in fluid communication with a liquid source,centrifuging the substrate about a central axis that is perpendicular tothe channels so that the liquid is dispersed from the central reservoirregion into said channels and chambers, such that any air bubbles in thechambers, channels, and/or passageways are forced towards the axis ofrotation, yielding a plurality of bubble-free electrophoretic pathsbetween said reservoir and said chambers.
 17. A method for preparing aplurality of electrophoretic paths which are substantially bubble-free,comprising providing an apparatus as defined in claim 1 such that thereservoir region, and optionally the channels, passageways, and/orchambers contain a liquid, centrifuging the substrate about a centralaxis that is perpendicular to the channels so that the liquid isdispersed from the central reservoir region into said channels andchambers, such that any air bubbles in the chambers, channels, and/orpassageways are forced towards the axis of rotation, yielding aplurality of bubble-free electrophoretic paths between said reservoirand said chambers.
 18. A method for analyzing a plurality of samplescomprising: providing an apparatus in accordance with claim 1, such thatthe central reservoir region, channels, and chambers contain a liquidmedium suitable for electrophoresis of such samples, applying anelectric field under conditions effective to cause migration of samplethrough at least one of said channels towards the central reservoirregion, and interrogating at least one of the channel(s) to detect oneor more sample components in the channel(s).
 19. The method of claim 18,wherein the components to be detected are nucleic acids.
 20. The methodof claim 19, wherein the nucleic acids to be detected are fluorescentlylabeled.
 21. The method of claim 19, wherein the samples are prepared bypolymerase chain reaction amplification of one or more selected targetsequences.
 22. The method of claim 19, wherein the samples are preparedby ligating at least two oligonucleotides when the oligonucleotides arebound to adjacent regions or a target polynucleotide that arecomplementary to the at least two oligonucleotides.
 23. The method ofclaim 18, wherein the components to be detected are polypeptides.